Peptides used for pharmaceutical purposes will, in the future, more frequently be produced through the exploitation of genetic engineering. However, genetic engineering has limits to its capabilities. For example, expression of recombinant peptides bearing non-naturally occurring L-amino acids, D-amino acids, radioactive amino acids, and other detectable labels is not possible through recombinant techniques because there is no genetic code which codes for these modifications. In addition, naturally occurring amino acid modifications such as C-terminal amide group substitution, which are routinely performed in vivo, are difficult to perform in vitro. These post-translation modifications are important because they often result in the most potent or longest acting form of the peptide and constitute the naturally occurring form of the peptide often needed for pharmaceutical use.
There are techniques for modification of recombinant peptides. One such technique is C-terminal .alpha.-carboxyl amidation, as described by Bongers et al., Int. J. Peptide Protein Res., 40:268 (1992) utilizing an .alpha. amidating enzyme as described in Henriksen et al., J. Am. Chem. Soc., 114:1876-1877 (1992); and Ohsuye et al., Biochem. Biophys. Res. Commun., 150:1275-1281 (1988). However, these techniques are limited to those modifications for which there exists a natural enzyme or chemical method capable of performing the desired modification.
Amidation of peptides has been performed through protease catalyzed replacement reactions (transpeptidation) using an amino acid amide or peptide amide as a nucleophile. Sahina et al., Chem. Pharm. Bull., 36:4345-4354 (1988); Sahina et al., Chem. Pharm. Bull., 37:811-812 (1989); Breddam et al., J. Peptide Protein Res., 37:153-160 (1991). Yields using these techniques are typically quite low. However, transpeptidation reactions catalyzed by serine or thiol-proteases, under appropriate reaction conditions, have been carried out in high yields. Breddam et al. (1991) cited supra. Although protease catalyzed transpeptidation can be very effective under some circumstances, it is limited to substrates for which a natural protease exists and which exhibits specificity for a peptide bond close to the C-terminus.
Hence, there is a need to provide mutant protease enzymes capable of performing heretofor unknown N- or C-terminal modifications as well as peptide chain elongation with a variety of substrates, especially those substrates that are not reactive with the naturally occurring protease enzyme.